Difference between revisions of "Part:BBa K808010"
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The original part BBa K808010 was obtained via colony PCR from its original host ''Comamonas testosteroni Kf-1''. This is a fast and easy approach to obtain a gene without having it synthesized. A drawback when using this approach is the variation of codon usage in different host organisms. This can cause problems in the protein expression when transferring the gene into a different host. To allow efficient work with TphB in ''Escherichia coli'' (''E. coli'') our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. With those improvements our team managed a very strong overexpression of tphB.<br> | The original part BBa K808010 was obtained via colony PCR from its original host ''Comamonas testosteroni Kf-1''. This is a fast and easy approach to obtain a gene without having it synthesized. A drawback when using this approach is the variation of codon usage in different host organisms. This can cause problems in the protein expression when transferring the gene into a different host. To allow efficient work with TphB in ''Escherichia coli'' (''E. coli'') our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. With those improvements our team managed a very strong overexpression of tphB.<br> | ||
| − | [[Image:BBa K4378999 TphB overex.PNG|500px|thumb|center| Figure 2. Overexpression at two different temperature and IPTG concentrations of TphB using pET24a expression vectors. IF3 was used as control for overexpression. The TphB band is very strong and spans over huge area the expected size is 33 kDa which corresponses to the lower end of the band. The IF3 band is expected to be around 23 kDa]] | + | [[Image:BBa K4378999 TphB overex.PNG|500px|thumb|center| Figure 2. Overexpression at two different temperature and IPTG concentrations of TphB using pET24a expression vectors. IF3 was used as control for overexpression. The TphB band is very strong and spans over huge area the expected size is 33 kDa which corresponses to the lower end of the band. The IF3 band is expected to be around 23 kDa.]] |
These improvements resulted in the basic part BBa_K4378999 and the composite part BBa_K4378000 which can be directly used for overexpression in a pET-Vector without the use of any other functional parts of the T7 expression cassette. | These improvements resulted in the basic part BBa_K4378999 and the composite part BBa_K4378000 which can be directly used for overexpression in a pET-Vector without the use of any other functional parts of the T7 expression cassette. | ||
Revision as of 15:33, 5 October 2022
tphB: reaction from DCD to protocatechuate
TphB is coding for the decarboxylating cis-dihydrodiol dehydrogenase from Comamonas testosteroni Kf-1. It catalyzes the second reaction in the terephthalic acid degration - the decarboxylation of DCD to protocatechuate. The [http://2012.igem.org/Team:TU_Darmstadt/Modeling_Homologie_Modeling#TphB homology modeling] suggests a homodimerization as described by the recent paper by Bains et al.
For further information, please visit the [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism TU Darmstadt iGEM team wiki].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 155
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 202
Illegal NgoMIV site found at 877
Illegal AgeI site found at 939 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 76
Part Characterisation
GPC confirmed the suggested homodimerization (Figure 1).
Uppsala 2022 improvement
The original part BBa K808010 was obtained via colony PCR from its original host Comamonas testosteroni Kf-1. This is a fast and easy approach to obtain a gene without having it synthesized. A drawback when using this approach is the variation of codon usage in different host organisms. This can cause problems in the protein expression when transferring the gene into a different host. To allow efficient work with TphB in Escherichia coli (E. coli) our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. With those improvements our team managed a very strong overexpression of tphB.
These improvements resulted in the basic part BBa_K4378999 and the composite part BBa_K4378000 which can be directly used for overexpression in a pET-Vector without the use of any other functional parts of the T7 expression cassette.
References
- Sasoh, M., E. Masai, et al. (2006). "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 72(3): 1825-1832.
- Fukuhara, Y., K. Inakazu, et al. (2010). "Characterization of the isophthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 76(2): 519-527.
- Kamimura, N., T. Aoyama, et al. (2010). "Characterization of the protocatechuate 4,5-cleavage pathway operon in Comamonas sp. strain E6 and discovery of a novel pathway gene." Appl Environ Microbiol 76(24): 8093-8101.
- Schläfli HR, Weiss MA, Leisinger T, Cook AM. (1994)"Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component." J Bacteriol. 1994 Nov;176(21):6644-52.
- Bains, J., J.E. Wulff, and M.J. Boulanger, Investigating Terephthalate Biodegradation: Structural Characterization of a Putative Decarboxylating cis-Dihydrodiol Dehydrogenase. J Mol Biol, 2012.